RESUMO
Magnetic resonance rotating frame relaxation times are an alternative non-contrast agent choice for the diagnosis of chronic myocardial infarct. Fibrosis typically occurs in progressive hypertrophic cardiomyopathy. Fibrosis has been imaged in myocardial infarcted tissue using rotating frame relaxation times, which provides the possibility to follow up progressive cardiomyopathy without contrast agents. Mild and severe left ventricular hypertrophy were induced in mice by transverse aortic constriction, and the longitudinal rotating frame relaxation times (T1ρ ) and relaxation along the fictitious field (TRAFF2 , TRAFF3 ) were measured at 5, 10, 24, 62 and 89 days after transverse aortic constriction in vivo. Myocardial fibrosis was verified using Masson's trichrome staining. Increases in the relative relaxation time differences of T1ρ , together with TRAFF2 and TRAFF3 , between fibrotic and remote tissues over time were observed. Furthermore, TRAFF2 and TRAFF3 showed higher relaxation times overall in fibrotic tissue than T1ρ . Relaxation time differences were highly correlated with an excess of histologically verified fibrosis. We found that TRAFF2 and TRAFF3 are more sensitive than T1ρ to hypertrophic cardiomyopathy-related tissue changes and can serve as non-invasive diagnostic magnetic resonance imaging markers to follow up the mouse model of progressive hypertrophic cardiomyopathy.
Assuntos
Cardiomiopatia Hipertrófica/diagnóstico por imagem , Cardiomiopatia Hipertrófica/patologia , Progressão da Doença , Imageamento por Ressonância Magnética , Rotação , Animais , Seguimentos , Masculino , Camundongos Endogâmicos C57BL , Miocárdio/patologia , Fatores de TempoRESUMO
AIMS: We evaluated for the first time the effects of angiogenic and lymphangiogenic AdVEGF-DΔNΔC gene therapy in patients with refractory angina. METHODS AND RESULTS: Thirty patients were randomized to AdVEGF-DΔNΔC (AdVEGF-D) or placebo (control) groups. Electromechanical NOGA mapping and radiowater PET were used to identify hibernating viable myocardium where treatment was targeted. Safety, severity of symptoms, quality of life, lipoprotein(a) [Lp(a)] and routine clinical chemistry were measured. Myocardial perfusion reserve (MPR) was assessed with radiowater PET at baseline and after 3- and 12-months follow-up. Treatment was well tolerated. Myocardial perfusion reserve increased significantly in the treated area in the AdVEGF-D group compared with baseline (1.00 ± 0.36) at 3 months (1.31 ± 0.46, P = 0.045) and 12 months (1.44 ± 0.48, P = 0.009) whereas MPR in the reference area tended to decrease (2.05 ± 0.69, 1.76 ± 0.62, and 1.87 ± 0.69; baseline, 3 and 12 months, respectively, P = 0.551). Myocardial perfusion reserve in the control group showed no significant change from baseline to 3 and 12 months (1.26 ± 0.37, 1.57 ± 0.55, and 1.48 ± 0.48; respectively, P = 0.690). No major changes were found in clinical chemistry but anti-adenovirus antibodies increased in 54% of the treated patients compared with baseline. AdVEGF-D patients in the highest Lp(a) tertile at baseline showed the best response to therapy (MPR 0.94 ± 0.32 and 1.76 ± 0.41 baseline and 12 months, respectively, P = 0.023). CONCLUSION: AdVEGF-DΔNΔC gene therapy was safe, feasible, and well tolerated. Myocardial perfusion increased at 1 year in the treated areas with impaired MPR at baseline. Plasma Lp(a) may be a potential biomarker to identify patients that may have the greatest benefit with this therapy.
Assuntos
Angina Pectoris/terapia , Terapia Genética/métodos , Fator D de Crescimento do Endotélio Vascular/administração & dosagem , Adenoviridae , Idoso , Estudos de Viabilidade , Feminino , Seguimentos , Reserva Fracionada de Fluxo Miocárdico/fisiologia , Técnicas de Transferência de Genes , Vetores Genéticos , Humanos , Masculino , Imagem de Perfusão do Miocárdio/métodos , Neovascularização Fisiológica/fisiologia , Qualidade de Vida , Resultado do Tratamento , Fator D de Crescimento do Endotélio Vascular/efeitos adversosRESUMO
Plasmid-based Sleeping Beauty (SB) transposon vectors were developed and used to deliver genes for low-density lipoprotein and very-low-density lipoprotein receptors (LDLR and VLDLR, respectively) or lacZ reporter into liver of an LDLR-deficient mouse model of familial hypercholesterolemia (FH). SB transposase, SB100x, was used to integrate the therapeutic transposons into mice livers for evaluating the feasibility of the vectors in reducing high blood cholesterol and the progression of atherosclerosis. Hydrodynamic gene delivery of transposon-VLDLR into the livers of the mice resulted in initial 17-19% reductions in plasma cholesterol, and at the later time points, in a significant stabilization of the cholesterol level for the 6.5-month duration of the study compared to the control mice. Transposon-LDLR-treated animals also demonstrated a trend of stabilization in the cholesterol levels in the long term. Vector-treated mice had slightly less lipid accumulation in the liver and reduced aortic atherosclerosis. Clinical chemistry and histological analyses revealed normal liver function and morphology comparable to that of the controls during the follow-up with no safety issues regarding the vector type, transgenes, or the gene transfer method. The study demonstrates the safety and potential benefits of the SB transposon vectors in the treatment of FH.
Assuntos
Elementos de DNA Transponíveis , Técnicas de Transferência de Genes , Vetores Genéticos/genética , Hiperlipoproteinemia Tipo II/genética , Fígado/metabolismo , Receptores de LDL/genética , Animais , Aorta/metabolismo , Aorta/patologia , Aterosclerose/metabolismo , Aterosclerose/patologia , Linhagem Celular , Modelos Animais de Doenças , Expressão Gênica , Ordem dos Genes , Terapia Genética , Hiperlipoproteinemia Tipo II/metabolismo , Hiperlipoproteinemia Tipo II/terapia , Lipídeos/sangue , Camundongos , Camundongos KnockoutRESUMO
BACKGROUND: Vascular endothelial growth factors (VEGFs) are potential therapeutic agents for treatment of ischemic diseases. Their angiogenic effects are mainly mediated through VEGF receptor 2 (VEGFR2). METHODS: Receptor binding, signaling, and biological efficacy of several VEGFR2 ligands were compared to determine their characteristics regarding angiogenic activity and vascular permeability. RESULTS: Tested VEGFR2 ligands induced receptor tyrosine phosphorylation with different efficacy depending on their binding affinities. However, the tyrosine phosphorylation pattern and the activation of the major downstream signaling pathways were comparable. The maximal angiogenic effect stimulated by different VEGFR2 ligands was dependent on their ability to bind to co-receptor Neuropilin (Nrp), which was shown to form complexes with VEGFR2. The ability of these VEGFR2 ligands to induce vascular permeability was dependent on their concentration and VEGFR2 affinity, but not on Nrp binding. CONCLUSIONS: VEGFR2 activation alone is sufficient for inducing endothelial cell proliferation, formation of tube-like structures and vascular permeability. The level of VEGFR2 activation is dependent on the binding properties of the ligand used. However, closely similar activation pattern of the receptor kinase domain is seen with all VEGFR2 ligands. Nrp binding strengthens the angiogenic potency without increasing vascular permeability. GENERAL SIGNIFICANCE: This study sheds light on how different structurally closely related VEGFR2 ligands bind to and signal via VEGFR2/Nrp complex to induce angiogenesis and vascular permeability. The knowledge of this study could be used for designing VEGFR2/Nrp ligands with improved therapeutic properties.
Assuntos
Aorta/metabolismo , Endotélio Vascular/metabolismo , Células Endoteliais da Veia Umbilical Humana/metabolismo , Neovascularização Fisiológica , Fator A de Crescimento do Endotélio Vascular/metabolismo , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/metabolismo , Animais , Aorta/citologia , Western Blotting , Permeabilidade Capilar , Movimento Celular , Proliferação de Células , Células Cultivadas , Endotélio Vascular/citologia , Células Endoteliais da Veia Umbilical Humana/citologia , Humanos , Imunoprecipitação , Fosforilação , Plasmídeos , Transdução de Sinais , Suínos , Fator A de Crescimento do Endotélio Vascular/genética , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/genéticaRESUMO
OBJECTIVE: Lymphatic vessels collect extravasated fluid and proteins from tissues to blood circulation as well as play an essential role in lipid metabolism by taking up intestinal chylomicrons. Previous studies have shown that impairment of lymphatic vessel function causes lymphedema and fat accumulation, but clear connections between arterial pathologies and lymphatic vessels have not been described. APPROACH AND RESULTS: Two transgenic mouse strains with lymphatic insufficiency (soluble vascular endothelial growth factor 3 [sVEGFR3] and Chy) were crossed with atherosclerotic mice deficient of low-density lipoprotein receptor and apolipoprotein B48 (LDLR(-/-)/ApoB(100/100)) to study the effects of insufficient lymphatic vessel transport on lipoprotein metabolism and atherosclerosis. Both sVEGFR3×LDLR(-/-)/ApoB(100/100) mice and Chy×LDLR(-/-)/ApoB(100/100) mice had higher plasma cholesterol levels compared with LDLR(-/-)/ApoB(100/100) control mice during both normal chow diet (16.3 and 13.7 versus 8.2 mmol/L, respectively) and Western-type high-fat diet (eg, after 2 weeks of fat diet, 45.9 and 42.6 versus 30.2 mmol/L, respectively). Cholesterol and triglyceride levels in very-low-density lipoprotein and low-density lipoprotein fractions were increased. Atherosclerotic lesions in young and intermediate cohorts of sVEGFR3×LDLR(-/-)/ApoB(100/100) mice progressed faster than in control mice (eg, intermediate cohort mice at 6 weeks, 18.3% versus 7.7% of the whole aorta, respectively). In addition, lesions in sVEGFR3×LDLR(-/-)/ApoB(100/100) mice and Chy×LDLR(-/-)/ApoB(100/100) mice had much less lymphatic vessels than lesions in control mice (0.33% and 1.07% versus 7.45% of podoplanin-positive vessels, respectively). CONCLUSIONS: We show a novel finding linking impaired lymphatic vessels to lipoprotein metabolism, increased plasma cholesterol levels, and enhanced atherogenesis.
Assuntos
Aterosclerose/etiologia , Hipercolesterolemia/complicações , Lipoproteínas/metabolismo , Vasos Linfáticos/fisiopatologia , Animais , Apolipoproteínas B/fisiologia , Colesterol/metabolismo , Humanos , Lipídeos/sangue , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Receptores de LDL/fisiologia , Receptor 3 de Fatores de Crescimento do Endotélio Vascular/fisiologiaRESUMO
BACKGROUND: The aim of the study was to explore the serum levels of eight angiogenesis biomarkers in patients with benign, borderline or malignant epithelial ovarian neoplasms and to compare them to those of healthy controls. In addition, we aimed to study how those biomarkers predict the clinical course and survival of patients with epithelial ovarian cancer. METHODS: We enrolled 132 patients with ovarian neoplasms and 32 unaffected women in this study. Serum samples were collected preoperatively at the time of diagnosis and the levels of angiogenesis biomarkers were measured with an ELISA. RESULTS: Levels of Ang-1, Ang-2, VEGF, VEGF-D, VEGF/sVEGFR-2 and Ang-2/ sVEGFR-2 ratios were elevated whereas sVEGFR-2 was lower in patients with ovarian carcinoma than in women with normal ovaries, benign and/or borderline ovarian neoplasms. In ROC analysis, the area under the curve for serum Ang-2/sVEGFR-2 ratio (0.76) was greater than Ang-2 (0.75) and VEGF (0.65) but lower than for CA 125 (0.90) to differentiate ovarian cancer from benign or borderline ovarian tumors. In ovarian cancer high Ang-2/sVEGFR-2 ratio was associated with the presence of ascites, high stage and grade of ovarian cancer, with the size of primary residual tumor>1 cm and with recurrence of disease. Elevated Ang-2, VEGF, VEGF/sVEGFR-2, Ang-2/VEGF and Ang-2/sVEGFR-2 ratios and low level of sVEGFR-2 were significant predictors of poor overall survival (OS) and recurrence free survival (RFS) in univariate survival analyses. CONCLUSIONS: Ovarian cancer patients had elevated levels of angiogenesis related growth factors in circulation reflecting increased angiogenesis and poor prognosis. The serum level of Ang-2 predicted most accurately poor OS and Ang-2/sVEGFR-2 ratio malignancy of ovarian neoplasms and short RFS.
Assuntos
Angiopoietina-2/sangue , Neoplasias Epiteliais e Glandulares/sangue , Neoplasias Epiteliais e Glandulares/patologia , Neoplasias Ovarianas/sangue , Neoplasias Ovarianas/patologia , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/sangue , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Área Sob a Curva , Biomarcadores/sangue , Antígeno Ca-125/sangue , Carcinoma Epitelial do Ovário , Estudos de Casos e Controles , Feminino , Humanos , Pessoa de Meia-Idade , Gradação de Tumores , Recidiva Local de Neoplasia , Neoplasias Epiteliais e Glandulares/mortalidade , Neovascularização Patológica/sangue , Neoplasias Ovarianas/mortalidade , Prognóstico , Adulto JovemRESUMO
Longitudinal relaxation time in the rotating frame (T1ρ) was measured using continuous wave irradiation in normal and infarcted mouse myocardium in vivo. Significant increase in T1ρ was found after 7 days of infarction when compared with reference myocardium or in myocardium before infarction. Cine MRI and histology were performed to verify the severity of infarction. The time course of T1ρ in the infarct fits better with granulation and scar tissue formation than necrosis and edema. The results of the study show that T1ρ could potentially be a noninvasive quantitative marker for tissue remodeling after ischemic damage.
Assuntos
Algoritmos , Interpretação de Imagem Assistida por Computador/métodos , Imagem Cinética por Ressonância Magnética/métodos , Ponte Miocárdica/patologia , Infarto do Miocárdio/patologia , Miocárdio/patologia , Técnica de Subtração , Animais , Feminino , Aumento da Imagem/métodos , Estudos Longitudinais , Camundongos , Camundongos Endogâmicos C57BL , Ponte Miocárdica/etiologia , Infarto do Miocárdio/complicações , Reprodutibilidade dos Testes , Rotação , Sensibilidade e EspecificidadeRESUMO
BACKGROUND: Occluded arteries and ischemic tissues cannot always be treated by angioplasty, stenting or by-pass-surgery. Under such circumstances, viral gene therapy may be useful in inducing increased blood supply to ischemic area. There is evidence of improved blood flow in ischemic skeletal muscle and myocardium in both animal and human studies using adenoviral vascular endothelial growth factor (VEGF) gene therapy. However, the expression is transient and repeated gene transfers with the same vector are inefficient due to immune responses. METHODS: Different baculoviral vectors pseudotyped with or without vesicular stomatitis virus glycoprotein (VSV-G) and/or carrying woodchuck hepatitis virus post-transcriptional regulatory element (Wpre) were tested both in vitro and in vivo. VEGF-D(ΔNΔC) was used as therapeutic transgene and lacZ as a control. In vivo efficacy was evaluated as capillary enlargement and transgene expression in New Zealand White (NZW) rabbit skeletal muscle. RESULTS: A statistically significant capillary enlargement was detected 6 days after gene transfer in transduced areas compared to the control gene transfers with baculovirus and adenovirus encoding ß-galactosidase (lacZ). Substantially improved gene transfer efficiency was achieved with a modified baculovirus pseudotyped with VSV-G and carrying Wpre. Dose escalation experiments revealed that either too large volume or too many virus particles caused inflammation and necrosis in the target tissue, whereas 10(9) plaque forming units injected in multiple aliquots resulted in transgene expression with only mild immune reactions. CONCLUSIONS: We show the first evidence of biologically significant baculoviral gene transfer in skeletal muscle of NZW rabbits using VEGF-D(ΔNΔC) as a therapeutic transgene.
Assuntos
Baculoviridae/genética , Técnicas de Transferência de Genes , Músculo Esquelético/irrigação sanguínea , Neovascularização Fisiológica/genética , Fator D de Crescimento do Endotélio Vascular/genética , Fator D de Crescimento do Endotélio Vascular/uso terapêutico , Animais , Capilares/crescimento & desenvolvimento , Permeabilidade Capilar , Feminino , Técnicas de Transferência de Genes/efeitos adversos , Células Hep G2 , Humanos , Músculo Esquelético/patologia , Perfusão , Molécula-1 de Adesão Celular Endotelial a Plaquetas/metabolismo , Isoformas de Proteínas/genética , Coelhos , Proteínas Recombinantes/biossíntese , Transdução Genética , Resultado do TratamentoRESUMO
Interferon alpha (IFNα) gene therapy is emerging as a new treatment option for patients with non-muscle invasive bladder cancer (NMIBC). Adenoviral vectors expressing IFNα have shown clinical efficacy treating bacillus Calmette-Guerin (BCG)-unresponsive bladder cancer (BLCA). However, transient transgene expression and adenoviral immunogenicity may limit therapeutic activity. Lentiviral vectors can achieve stable transgene expression and are less immunogenic. In this study, we evaluated lentiviral vectors expressing murine IFNα (LV-IFNα) and demonstrate IFNα expression by transduced murine BLCA cell lines, bladder urothelium, and within the urine following intravesical instillation. Murine BLCA cell lines (MB49 and UPPL1541) were sensitive to IFN-mediated cell death after LV-IFNα, whereas BBN975 was inherently resistant. Upregulation of interleukin-6 (IL-6) predicted sensitivity to IFN-mediated cell death mediated by caspase signaling, which when inhibited abrogated IFN-mediated cell killing. Intravesical therapy with LV-IFNα/Syn3 in a syngeneic BLCA model significantly improved survival, and molecular analysis of treated tumors revealed upregulation of apoptotic and immune-cell-mediated death pathways. In particular, biomarker discovery analysis identified three clinically actionable targets, PD-L1, epidermal growth factor receptor (EGFR), and ALDHA1A, in murine tumors treated with LV-IFNα/Syn3. Our findings warrant the comparison of adenoviral and LV-IFNα and the study of novel combination strategies with IFNα gene therapy for the BLCA treatment.
RESUMO
Biological bypass through induced angiogenesis by vascular endothelial growth factor D (VEGF-D) gene therapy (GT) is a new concept for the treatment of cardiac ischemia. Serotype 5 adenoviruses are used in the clinical trials for transferring the VEGF-D cDNA into the ischemic myocardium. However, the presence of replication-competent vectors in the adenovirus products is a widely recognized problem that may pose a potential safety risk to the treated patients. We compared three different VEGF-D GT production lots containing different levels of replication-competent adenoviruses (RCA) tested in 3 × 1010 viral particles (vp): <10 RCA (VEGF-D L-RCA1), 10-100 RCA (VEGF-D H-RCA2), and 100-200 RCA (VEGF-D H-RCA3), as measured by a novel droplet digital polymerase chain reaction (PCR) RCA assay in a preclinical rabbit model (n = 21). ß-galactosidase encoding nonclinical-grade preparation was used as a nonangiogenic control. Each preparation was injected into the right semimembranosus muscle using dose of 1 × 1011 vp. Efficacy of the products was tested by the combination of contrast pulse sequencing ultrasound and modified Miles assay as well as quantifying the total cross-sectional area of capillaries. Safety, immunogenicity, toxicity, biodistribution, and shedding were assessed by general histology, serial measurements of C-reactive protein, white blood cell count and body temperature as well as using quantitative real-time PCR with primers targeted to the VEGF-D and replication-permitting E1 sequences. We found no significant differences in the efficacy or safety between the study groups. Most importantly, no detectable presence of RCA-specific E1 sequence was found in any samples tested, indicating that no detectable vector replication took place in vivo. We conclude that relatively low levels of RCA in adenoviral GT products may not be as important major safety issue as previously anticipated.
Assuntos
Adenovírus Humanos , Fator D de Crescimento do Endotélio Vascular , Adenoviridae/genética , Adenoviridae/metabolismo , Animais , Terapia Genética , Vetores Genéticos/genética , Humanos , Neovascularização Patológica , Coelhos , Distribuição Tecidual , Fator D de Crescimento do Endotélio Vascular/genética , Fator D de Crescimento do Endotélio Vascular/metabolismoRESUMO
BACKGROUND: New revascularization therapies are urgently needed for patients with severe coronary heart disease who lack conventional treatment options. METHODS AND RESULTS: We describe a new proangiogenic approach for these no-option patients using adenoviral (Ad) intramyocardial vascular endothelial growth factor (VEGF)-B186 gene transfer, which induces myocardium-specific angiogenesis and arteriogenesis in pigs and rabbits. After acute infarction, AdVEGF-B186 increased blood vessel area, perfusion, ejection fraction, and collateral artery formation and induced changes toward an ischemia-resistant myocardial phenotype. Soluble VEGF receptor-1 and soluble neuropilin receptor-1 reduced the effects of AdVEGF-B186, whereas neither soluble VEGF receptor-2 nor inhibition of nitric oxide production had this result. The effects of AdVEGF-B186 involved activation of neuropilin receptor-1, which is highly expressed in the myocardium, via recruitment of G-protein-alpha interacting protein, terminus C (GIPC) and upregulation of G-protein-alpha interacting protein. AdVEGF-B186 also induced an antiapoptotic gene expression profile in cardiomyocytes and had metabolic effects by inducing expression of fatty acid transport protein-4 and lipid and glycogen accumulation in the myocardium. CONCLUSIONS: VEGF-B186 displayed strikingly distinct effects compared with other VEGFs. These effects may be mediated at least in part via a G-protein signaling pathway. Tissue-specificity, high efficiency in ischemic myocardium, and induction of arteriogenesis and antiapoptotic and metabolic effects make AdVEGF-B186 a promising candidate for the treatment of myocardial ischemia.
Assuntos
Artérias/efeitos dos fármacos , Isquemia Miocárdica/terapia , Neovascularização Fisiológica/efeitos dos fármacos , Neuropilina-1/metabolismo , Fator B de Crescimento do Endotélio Vascular/administração & dosagem , Receptor 1 de Fatores de Crescimento do Endotélio Vascular/metabolismo , Animais , Artérias/crescimento & desenvolvimento , Terapia Genética/métodos , Vetores Genéticos , Infarto do Miocárdio/terapia , Traumatismo por Reperfusão Miocárdica/prevenção & controle , Especificidade de Órgãos , Coelhos , SuínosRESUMO
Human 15-lipoxygenase-1 (15-LO-1) is an oxidizing enzyme capable of producing reactive lipid hydroperoxides. 15-LO-1 and its products have been suggested to be involved in many pathological conditions, such as inflammation, atherogenesis, and carcinogenesis. We used adenovirus-mediated gene transfers to study the effects of 15-LO-1 on vascular endothelial growth factor (VEGF)-A165- and placental growth factor (PlGF)-induced angiogenesis in rabbit skeletal muscles. 15-LO-1 significantly decreased all angiogenic effects induced by these growth factors, including capillary perfusion, vascular permeability, vasodilatation, and an increase in capillary number. The effects are attributable to the reduction in the amount of VEGF-A165 and PlGF transcripts by 15-LO-1, resulting in reduced protein expression. The most likely mediator of the VEGF family-induced capillary vasodilatation is nitric oxide (NO), which is produced by NO synthases. Endothelial NO synthase protein expression and NO synthase activity were significantly induced by VEGF-A165, and these inductions were reduced by 15-LO-1. VEGF-A165 induces its angiogenic effects primarily via vascular endothelial growth factor receptor (VEGFR)2, and also PlGF mediates angiogenic signaling via VEGFR2, even though it binds to VEGFR1. VEGFR2 expression is induced by peroxisome proliferator-activating receptor . We showed by quantitative RT-PCR and immunohistochemistry that expression of endogenous rabbit peroxisome proliferator-activating receptor and VEGFR2 were significantly increased in the growth factor-transduced muscles, but these inductions were efficiently prevented by 15-LO-1. In conclusion, the results suggest that expression of 15-LO-1 has an efficient antiangiogenic effect in vivo via reduction in growth factor mRNA levels, NO bioactivity, and VEGFR2 expression.
Assuntos
Araquidonato 15-Lipoxigenase/fisiologia , Músculo Esquelético/irrigação sanguínea , Neovascularização Fisiológica , Proteínas da Gravidez/fisiologia , Fator A de Crescimento do Endotélio Vascular/fisiologia , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/genética , Animais , Araquidonato 15-Lipoxigenase/genética , Regulação para Baixo/genética , Técnicas de Transferência de Genes , Humanos , Camundongos , Óxido Nítrico/metabolismo , Fator de Crescimento Placentário , Proteínas da Gravidez/genética , RNA Mensageiro/sangue , Coelhos , Fator A de Crescimento do Endotélio Vascular/genéticaRESUMO
INTRODUCTION: The aims of the study were to explore the levels of angiopoietin-1 (Ang-1) and angiopoietin-2 (Ang-2) in patients with benign, borderline, or malignant epithelial ovarian tumors and to compare them to those of healthy controls. In addition, we aimed to study how Ang-1 and Ang-2 levels predict the clinical course and survival of patients with epithelial ovarian cancer. METHODS: We enrolled 150 patients with ovarian neoplasms and 34 women with healthy ovaries in this study. Furthermore, we measured the levels of Ang-1 and Ang-2 in patients having an ovarian metastasis or another cancer (n = 29). Serum samples were collected preoperatively at the time of diagnosis, and Ang-1 and Ang-2 levels were measured with an enzyme-linked immunosorbent assay. RESULTS: Angiopoietin-1 and Ang-2 levels were significantly elevated in serum samples of patients with ovarian carcinoma compared with healthy controls (P = 0.0005 and P < 0.0005, respectively). In addition, Ang-2 levels were significantly higher in patients with ovarian carcinoma compared with patients with benign (P < 0.0005) or borderline ovarian tumors (P = 0.011). In receiver operating characteristic analysis, the area under the curve for serum Ang-2 (0.77) was greater than Ang-1 (0.60) but lower than for cancer antigen 125 (0.95) to differentiate ovarian cancer from healthy control. High serum levels of Ang-1 and Ang-2 were associated with primary residual tumor more than 1 cm after debulking surgery, and high Ang-2 levels correlated positively with an advanced tumor stage (P = 0.042). Elevated Ang-2 level (>2.7 ng/mL) was a significant predictor of poor overall and recurrence-free survival (P = 0.043 and P = 0.033, respectively) when assessing Kaplan-Meier curves by a log-rank test. CONCLUSIONS: Patients with ovarian cancer have higher serum levels of angiopoietins than patients with benign or borderline tumors reflecting the increased angiogenesis. These results also suggest that Ang-2 may serve as an angiogenic marker of decreased patient survival in ovarian cancer.
Assuntos
Angiopoietina-2/sangue , Carcinoma/diagnóstico , Estadiamento de Neoplasias/métodos , Neoplasias Ovarianas/diagnóstico , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Angiopoietina-1/análise , Angiopoietina-1/sangue , Angiopoietina-2/análise , Biomarcadores Tumorais/análise , Biomarcadores Tumorais/sangue , Carcinoma/sangue , Carcinoma/mortalidade , Carcinoma/cirurgia , Carcinoma Epitelial do Ovário , Estudos de Casos e Controles , Feminino , Humanos , Pessoa de Meia-Idade , Neoplasias Epiteliais e Glandulares/sangue , Neoplasias Epiteliais e Glandulares/diagnóstico , Neoplasias Epiteliais e Glandulares/mortalidade , Neoplasias Epiteliais e Glandulares/cirurgia , Neoplasias Ovarianas/sangue , Neoplasias Ovarianas/mortalidade , Neoplasias Ovarianas/cirurgia , Período Pré-Operatório , Prognóstico , Análise de Sobrevida , Adulto JovemRESUMO
We studied antiangiogenic and antilymphangiogenic effects of sVEGFR-1 (sFlt-1), sVEGFR-2 (sFlk-1/KDR), and sVEGFR-3 (sFlt-4) gene transfers and their combinations in intraperitoneal ovarian cancer xenograft mice (Balb/c-Anu, n = 55). Gene therapy was initiated when the presence of sizable tumors was confirmed in magnetic resonance imaging (MRI). Adenovirus-mediated gene transfer was performed intravenously via tail vein as follows: AdLacZ as a control (group I), AdsFlt-1 (group II), AdsKDR (group III), AdsFlt-4 (group IV) and two combination groups of AdsFlt-1 and AdsFlt-4 (group V) and AdsFlt-1, AdsKDR, and AdsFlt-4 (group VI). Antitumor effectiveness was assessed by sequential MRI, immunohistochemistry, microvessel density, overall tumor growth, and survival time. In combination group VI, intraperitoneal tumors were significantly smaller than in the control group at the end of the follow-up (P < 0.001). Furthermore, in group VI the microvessel density (microvessels/mm(2)) in tumor tissue and the total area of tumors covered by microvessels were significantly smaller than in the controls. One mouse in group V was cured. The combined antiangiogenic gene therapy with soluble VEGFRs reduced tumor growth, tumor vascularity, and ascites formation in ovarian cancer xenografts. The results suggest that the combined antiangiogenic gene therapy is a potential approach for the treatment of ovarian cancer patients.
Assuntos
Terapia Genética/métodos , Neoplasias Ovarianas/terapia , Receptor 1 de Fatores de Crescimento do Endotélio Vascular/fisiologia , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/fisiologia , Receptor 3 de Fatores de Crescimento do Endotélio Vascular/fisiologia , Animais , Linhagem Celular Tumoral , Ensaio de Imunoadsorção Enzimática , Feminino , Vetores Genéticos , Humanos , Imuno-Histoquímica , Imageamento por Ressonância Magnética , Camundongos , Camundongos Nus , Neoplasias Ovarianas/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Receptor 1 de Fatores de Crescimento do Endotélio Vascular/genética , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/genética , Receptor 3 de Fatores de Crescimento do Endotélio Vascular/genéticaRESUMO
We have previously produced viral vectors (lentiviral vector, adenoviral vector, and adeno-associated viral vector) in small and in commercial scale in adherent cells using Pall fixed-bed iCELLis® bioreactor. Recently, a company called Univercells has launched a new fixed-bed bioreactor with the same cell growth surface matrix material, but with different fixed-bed structure than is used in iCELLis bioreactor. We sought to compare the new scale-X™ hydro bioreactor (2.4 m2) and iCELLis Nano system (2.67 m2) to see if the difference has any effect on cell growth or lentiviral vector and adenoviral vector productivity. Runs were performed using parameters optimized for viral vector production in iCELLis Nano bioreactor. Cell growth was monitored by counting nuclei, as well as by following glucose consumption and lactate production. In both bioreactor systems, cells grew well, and the cell distribution was found quite homogeneous in scale-X bioreactor. Univercells scale-X bioreactor was proven to be at least equally efficient or even improved in both lentiviral vector and adenoviral vector production. Based on the results, the same protocol and parameters used in viral vector production in iCELLis bioreactor can also be successfully used for the production in scale-X bioreactor system.
Assuntos
Adenoviridae/metabolismo , Vetores Genéticos/biossíntese , Lentivirus/metabolismo , Cultura de Vírus/métodos , Adenoviridae/crescimento & desenvolvimento , Reatores Biológicos , Terapia Genética , Células HEK293 , Células HeLa , Humanos , Lentivirus/crescimento & desenvolvimentoRESUMO
The interest in lentiviral vectors (LVs) has increased prominently for gene therapy applications, but few have reached the later stages of clinical trials. The main challenge has remained in scaling up the manufacturing process for the fragile vector to obtain high titers for in vivo usage. We have previously scaled up the LV production to iCELLis 500, being able to produce up to 180 L of harvest material in one run with perfusion. The following challenge considers the purification and concentration of the product to meet titer and purity requirements for clinical use. We have developed a downstream process, beginning with clarification, buffer exchange, and concentration, by tangential flow filtration. This is followed by a purification step using single membrane-based anion exchange chromatography and final formulation with tangential flow filtration. Different materials and conditions were compared to optimize the process, especially for the chromatography step that has been the bottleneck in lentiviral vector purification scale-up. The final infectious titer of the lentiviral vector product manufactured using the optimized scale-up process was determined to be 1.97 × 109 transducing units (TU)/mL, which can be considered as a high titer for lentiviral vectors.
RESUMO
Severe fetal growth restriction (FGR) affects 1:500 pregnancies, is untreatable and causes serious neonatal morbidity and death. Reduced uterine blood flow (UBF) and lack of bioavailable VEGF due to placental insufficiency is a major cause. Transduction of uterine arteries in normal or FGR sheep and guinea pigs using an adenovirus (Ad) encoding VEGF isoforms A (Ad.VEGF-A165) and a FLAG-tagged pre-processed short form D (DΔNΔC, Ad.VEGF-DΔNΔC-FLAG) increases endothelial nitric oxide expression, enhances relaxation and reduces constriction of the uterine arteries and their branches. UBF and angiogenesis are increased long term, improving fetal growth in utero. For clinical trial development we compared Ad.VEGF vector transduction efficiency and function in endothelial cells (ECs) derived from different species. We aimed to compare the transduction efficiency and function of the pre-clinical study Ad. constructs (Ad.VEGF-A165, Ad.VEGF-DΔNΔC-FLAG) with the intended clinical trial construct (Ad.VEGF-DΔNΔC) where the FLAG tag is removed. We infected ECs from human umbilical vein, pregnant sheep uterine artery, pregnant guinea pig aorta and non-pregnant rabbit aorta, with increasing multiplicity of infection (MOI) for 24 or 48 hours of three Ad.VEGF vectors, compared to control Ad. containing the LacZ gene (Ad.LacZ). VEGF supernatant expression was analysed by ELISA. Functional assessment used tube formation assay and Erk-Akt phosphorylation by ELISA. VEGF expression was higher after Ad.VEGF-DΔNΔC-FLAG and Ad.VEGF-DΔNΔC transduction compared to Ad.VEGF-A165 in all EC types (*p < 0.001). Tube formation was higher in ECs transduced with Ad.VEGF-DΔNΔC in all species compared to other constructs (***p < 0.001, *p < 0.05 with rabbit aortic ECs). Phospho-Erk and phospho-Akt assays displayed no differences between the three vector constructs, whose effect was, as in other experiments, higher than Ad.LacZ (***p < 0.001). In conclusion, we observed high transduction efficiency and functional effects of Ad.VEGF-DΔNΔC vector with comparability in major pathway activation to constructs used in pre-clinical studies, supporting its use in a clinical trial.
Assuntos
Adenoviridae/genética , Dependovirus/genética , Endotélio Vascular/metabolismo , Terapia Genética/métodos , Vetores Genéticos/administração & dosagem , Insuficiência Placentária/terapia , Fator A de Crescimento do Endotélio Vascular/genética , Animais , Feminino , Vetores Genéticos/genética , Cobaias , Células Endoteliais da Veia Umbilical Humana , Humanos , Insuficiência Placentária/genética , Insuficiência Placentária/metabolismo , Insuficiência Placentária/patologia , Gravidez , Coelhos , OvinosRESUMO
BACKGROUND: The present study aimed to determine the efficiency and safety of baculovirus-mediated intravitreal gene transfer in rabbit eye and to compare its efficiency with adenovirus. We also studied how an intravitreal injection of vectors producing vascular endothelial growth factor D (VEGF-D) impacts the vasculature of rabbit eye. METHODS: Baculoviral (BacVEGF-D) or adenoviral VEGF-D (AdVEGF-D) were administered intravitreally into the right eye at different doses (10(8), 10(9) and 10(10) IU/ml) to 24 animals. Left eyes were injected with control viruses. To determine how long transgene expression lasted, we injected BacVEGF-D or BacLacZ to the vitreous humour of 11 animals and followed them for 4 weeks. Vitreous samples were taken after sacrifice for enzyme-linked immunosorbent assays and eyes were removed and fixed for histological analyses. RESULTS: Both baculoviruses and adenoviruses caused efficient expression of VEGF-D in the rabbit eyes. BacVEGF-D caused a dose-dependent vascular leakage and a moderate dilation of the capillaries. The highest effect was seen 6 days after gene transfer and was detectable for 2 weeks. Intravitreal injection of baculovirus caused expression of VEGF-D in the inner retina, photoreceptor cells and in retinal pigment epithelium cells, whereas adenovirus-mediated VEGF-D expression was detected in the nerve fiber layer and ganglion cell layer. Baculovirus caused a transient inflammation similar to adenoviruses. CONCLUSIONS: The study suggests that baculoviruses are efficient vectors for ocular gene transfer, especially if deeper retinal layers need to be transduced. In addition, intravitreal VEGF-D gene transfer caused blood-retina barrier breakdown but not neovessel formation in the rabbit eye.
Assuntos
Adenoviridae/genética , Baculoviridae/genética , Vetores Genéticos/genética , Transdução Genética , Fator D de Crescimento do Endotélio Vascular/genética , Corpo Vítreo/metabolismo , Angiografia , Animais , Capilares/patologia , Ensaio de Imunoadsorção Enzimática , Fluoresceína , Inflamação/patologia , Injeções , Coelhos , Retina/patologia , Fatores de Tempo , Corpo Vítreo/irrigação sanguínea , Corpo Vítreo/patologiaRESUMO
Vascular endothelial growth factors regulate vascular and lymphatic growth. Dysregulation of VEGF signaling is connected to many pathological states, including hemangiomas, arteriovenous malformations and placental abnormalities. In heart, VEGF gene transfer induces myocardial angiogenesis. Besides vascular and lymphatic endothelial cells, VEGFs affect multiple other cell types. Understanding VEGF biology and its paracrine signaling properties will offer new targets for novel treatments of several diseases.
Assuntos
Malformações Arteriovenosas/metabolismo , Células Endoteliais/metabolismo , Cardiopatias/metabolismo , Hemangioma/metabolismo , Miocárdio/metabolismo , Placenta/metabolismo , Fator A de Crescimento do Endotélio Vascular/metabolismo , Animais , Malformações Arteriovenosas/genética , Malformações Arteriovenosas/patologia , Células Endoteliais/patologia , Feminino , Cardiopatias/patologia , Hemangioma/genética , Hemangioma/patologia , Humanos , Linfangiogênese , Miocárdio/patologia , Neovascularização Patológica , Neovascularização Fisiológica , Comunicação Parácrina , Placenta/patologia , Gravidez , Transdução de Sinais , Fator A de Crescimento do Endotélio Vascular/genéticaRESUMO
BACKGROUND: Adenoviral gene therapy, based on herpes simplex virus thymidine kinase (AdvTK) is being developed for clinical use but no safety data are available with respect to the effects on female germ cells should the virus accidentally be released into systemic circulation. We studied the effects of AdvTK gene therapy on ovaries and germ cells in pregnant and nonpregnant rabbits and also the potential transmission of a transgene to offspring. METHODS: To mimic the severest of conditions, gene transfer was made by direct catheter-mediated injection into the uterine artery of pregnant and nonpregnant rabbits. AdvTK or AdvLacZ at 1 x 10(10) pfu were used for gene transfer. Ganciclovir was administered to AdvTK-treated rabbits to induce gene therapy. The rabbits were mated 6 and 12 weeks following gene transfer and the surviving young (89 from a total of 114) were analysed. RESULTS: No change in fertility was observed in the two matings after the gene transfer. In addition, no change was observed in ovarian histology between the AdvTK group, the AdvLacZ group and the nontreated controls. Southern blotting analysis showed no genomic integration of the transgene. However, in PCR analysis, transgene DNA was found in 9.3% of the litter samples. This was not the case for results from the reverse transcription-PCR assay. CONCLUSIONS: Although AdvTK gene therapy may initially affect ovarian cells, the influence appears to be transient. However, after direct exposure of the ovarian cells in high concentration of adenoviruses, transmission of a transgene in the offspring cannot be excluded.